Glycopeptides and uses thereof

ABSTRACT

The present invention provides glycopeptides comprising a peptide that is covalently linked to a saccharide. The peptide portion of the glycopeptides of the invention has from about 20 to about 40 amino acid residues and at least 75% sequence identity to SEQ ID NO:1, 2, or 3. The saccharide moiety portion of the glycopeptides of the present invention comprises from 1 to about 8 carbohydrates. The present invention also relates to using the glycopeptides of the invention in treating various neurodegenerative diseases.

CROSS-REFERENCE TO RELATED APPLICATIONS

This application is a continuation-in-part (CIP) application of U.S.patent application Ser. No. 15/044,924, filed Feb. 16, 2016, which is aCIP application of the US National Phase of PCT Application No.PCT/US2014/051143 filed Aug. 14, 2014, which claims the priority benefitof U.S. Provisional Application Ser. No. 61/865,958, filed Aug. 14,2013, all of which are incorporated herein by reference in theirentirety.

STATEMENT REGARDING FEDERALLY FUNDED RESEARCH

This invention was made with Government support under Grant No.CHE-9526909 awarded by the National Science Foundation, Grant No.R01NS52727 and Grant No. R01NS091238, both awarded by the Department ofHealth and Human Services, National Institutes of Health, NationalInstitute of Neurological Disorders and Stroke. The Government hascertain rights in the invention.

FIELD OF THE INVENTION

The present invention relates to glycopeptides comprising a peptide thatis covalently linked to a saccharide. In particular, the peptide portionof the glycopeptides of the invention has from about 20 to about 40amino acid residues and at least 75% sequence identity to SEQ ID NO:1,2, or 3. The saccharide moiety portion of the glycopeptides of thepresent invention comprises from 1 to about 8 carbohydrates. The presentinvention also relates to using the glycopeptides of the invention intreating various neurodegenerative diseases. In particular,glycopeptides of the invention are useful in treating amyotrophiclateral sclerosis (ALS), Parkinson's Disease (PD), Alzheimer's Disease(AD), Huntington's Disease (HD), migraine attacks, traumatic braininjury and stroke, as well as certain forms of dementia.

BACKGROUND OF THE INVENTION

Endogenous opioid peptides, lumped together under the generic termendorphins, have been the subject of intense study since theirdiscovery. Neuropeptides have the potential for extremely selectivepharmacological intervention with fewer side effects. If these naturallyoccurring opioid peptides and their derivatives could be renderedpermeable to the blood-brain barrier (BBB), then a new vista ofpsychopharmacology would be opened to exploration and exploitation.After three decades of research, many potent and selective opioidagonists have been developed, and stability problems have been largelyovercome. The remaining problem that prevents the use of opioid peptidesas drugs is poor bioavailability, which is primarily due to poorpenetration of the BBB. The BBB is composed of endothelial cells in thecerebrovascular capillary beds. The BBB acts as a lipophilic barrier toundesired chemical substances, and admits vital nutrients for properfunction of the CNS. The flow is bi-directional, allowing for export ofmaterials from the CNS (efflux transport) and the import of materialsfrom the blood (influx transport). The BBB represents not only aphysical obstacle, but a metabolic one as well, possessing bothoxidative enzymes and peptidases such as aminopeptidase, arylamidase andenkephalinase. Thus, metabolically unstable substances (e.g. peptides)are generally degraded before they reach the CNS. It should also benoted that entry to the CNS does not guarantee that a drug willaccumulate in useful concentrations, as many peptides are rapidlyexported back to the bloodstream. Several strategies have been reportedto overcome the BBB penetration problem, including substitution ofunnatural amino acids, the use of conformational constraints, and theaddition of lipophilic side chains or other transport vectors.

Glycosylation has proven to be a successful methodology to improve boththe stability and bioavailability of short peptide “messages” byincorporation of the peptide pharmacophore into a glycopeptide. PreviousBBB penetration studies with opioid glycopeptide agonists based onenkephalins have shown up to 3-fold increases in the rate of braindelivery of these compounds compared with the unglycosylated parentpeptides. Recent studies with glycopeptides in artificial membranesystems indicate that amphipathicity of the glycopeptides is animportant factor in BBB penetration. In addition, there is evidence thatsuggests that the type of glycosylation can alter tissue distributionpatterns, BBB penetration and peptide/receptor interactions.

The endogenous neuropeptide ß-endorphin is a 31 residue naturallyoccurring opioid peptide agonist that binds to _(I)I. and 6 receptors.Its N-terminal 5 residues are identical to the Met-Enkephalin sequence,and may be considered to be the pharmacophore or “opioid message.” Itwas shown some time ago that the C-terminal region of ß-endorphin has anamphipathic α-helical structure that plays a role in the receptorbinding and opioid agonism and may induce resistance to proteolysis.Studies have shown the N-terminal sequence is the essential “message,”and the C-terminal helical region is the “address” that limits deliveryof the message to a subset of otherwise available opioid receptors. Somehave proposed that ß-endorphin consists of the Met-enkephalin peptidesequence at the N-terminus, a hydrophilic linker region from residues 6through 12, and an amphiphilic helical region between the helix breakerresidues Pro(13) and Gly(30). This was later proven by synthesizing anumber of ß-endorphin mimics with artificial C-terminal helical regionswith amphipathic character. These de novo amphipathic helices were nothomologous with the ß-endorphin C-terminal region, and they were shownto be largely α-helical by circular dichroism (CD) measurements. Thesehybrid structures showed good opioid agonism in vitro when compared toß-endorphin. These studies strongly suggested that the overallamphipathicity of the C-terminal helix plays a key role in theselectivity of these compounds, rather than the identity of specificamino acid residues present in the C-terminal.

Dynorphin A (1-17) is also an endogenous opioid peptide, but it bindspreferentially to the κ opioid receptor and has an N-terminal messagesegment identical to Leu-Enkephalin. It has been suggested that anaddress sequence in the C-terminal region imparts selectivity for κreceptors. Dynorphin A displayed an extended and/or random coilstructure when subjected to structural analysis by various spectroscopicmeasurements. A 2D (1) H-NMR study in DPC micelle showed that DynorphinA(1-17) contains a less ordered N-terminal segment, a well defineda-helix segment spanning between Phe(4) and Pro(10) or Lys(11), and aß-turn from Trp(14) to Gln(17). Based on NMR results, some believe thatboth the α-helix and the C-terminal ß-turn are due to dynorphin-micelleinteractions, and may be important structural features of thefull-length peptide when bound to the cell membrane in vivo.

Studies by others also support the notion that a helical structure inthe message segment of Dynorphin A(1-17) is significant. The biologicalimportance of helical C-terminal address segments in larger opioidpeptides has been further supported by the recent work by Kyle andco-workers. They successfully synthesized several potent nociceptin (NC)peptide analogs exploiting the α-helix-promoting residuesa-aminoisobutyric acid (Aib) and N-methyl alanine (MeAla) at theC-terminus of NC.

Nociceptin is the endogenous ligand for the recently identified opioidreceptor-like 1 receptor (ORL-1). Thus, it seems logical to approach thedesign of opioid agonist ß-endorphin or dynorphin peptide analogs bycombining C-terminal amphipathic helical address segments that can alsopromote BBB, for penetration by virtue of glycosylation.

It is an object of the present invention to provide glycopeptides thatcan readily penetrate the blood-brain-barriers (BBB) for treatment ofneurodegenerative diseases such as amyotrophic lateral sclerosis (ALS),Parkinson's Disease (PD), Alzheimer's Disease (AD), Huntington's Disease(HD), migraine attacks, traumatic brain injury and stroke, as well ascertain forms of dementia.

SUMMARY OF THE INVENTION

One aspect of the invention provides a glycopeptide comprising a peptidethat is covalently linked to a saccharide, wherein said peptide has fromabout 20 to about 40 amino acid residues and at least 75% sequenceidentity to pleiotropic peptide pituitary adenylate cyclase-activatingpolypeptide (PACAP) of SEQ ID NOs:1 or 2, or vasoactive intestinalpeptide (VIP) of SEQ ID NO:3, and wherein said saccharide comprises from1 to about 8 carbohydrates.

In one particular embodiment, the glycopeptide is selected from thegroup consisting of PACAP₁₋₂₇, PACAP₁₋₃₈, VIP, [AC-His¹]PACAP-27,[Ala²]PACAP-27, [Gly²⁰]PACAP-27, and Ac-[Phe(pI)⁶, Nle¹⁷]-PACAP₁₋₂₇.Within this embodiment, the glycopeptide is [Ala²]PACAP-27 is a D-isomeralanine, i.e., [D-Ala²]PACAP-27.

Still in another embodiment, at least one of the amino acid residues ofSEQ ID NOs:1, 2 or 3 is substituted with a substitution amino acidresidue having a side-chain functional group that is glycosylated.Typically, the substitution amino acid residue comprises serine,threonine, hydroxyproline or a similar ethanolamine linker.

In some embodiments, the glycopeptide has from about 25 to about 40,typically from about 25 to about 35, often about 25 to about 30, andmore often 27 to 29 amino acid residues.

Yet in other embodiments, the glycopeptide has at least 80%, typically85%, and often 90% sequence identity to PAPAC₁₋₂₇ (SEQ ID NO:2).

In yet other embodiments, the C-terminus end of the peptide isglycosylated.

Still yet in other embodiments, the glycopeptide is a pituitaryadenylate cyclase-activating polypeptide type I receptor (PAC₁) agonist.In other embodiments, the glycopeptide is a VPAC₁ agonist. Yet in otherembodiments, the glycopeptide is a selective PAC₁ and VPAC₁ agonist.

Yet in other embodiments, the PAC1 binding affinity (Ki) of theglycopeptide is less than about 10 nM.

In other embodiments, the VPAC1 binding affinity (Ki) of theglycopeptide is less than about 10 nM.

Still in other embodiments, the PAC1 agonist activity (Ki) of theglycopeptide is less than about 10 nM.

Yet in other embodiments, the VPAC1 agonist activity (κi) of saidglycopeptide is less than about 10 nM.

Typically, the saccharide comprises from 1 to 5, often 1 to 3carbohydrates. In one particular embodiment, the saccharide is amonosaccharide, a disaccharide.

Still yet in other embodiments, the peptide comprises a plurality ofglycosylated amino acid residues, i.e., different amino acids areglycosylated where each saccharide linked to the amino acid residue isindependently selected. In some embodiments, 1 to 5, typically 1 to 3,often 1 or 2 different amino acid residues are glycosylated. Often oneamino acid residue is glycosylated.

In some embodiments, the saccharide is selected from the groupconsisting of glucose, maltose, lactose, melibiose, maltotriose,sucrose, trehalose, altose, saccharose, maltose, cellobiose, gentibiose,isomaltose, primeveose, galactose, xylose, mannose, manosaminic acid,fucose, GalNAc, GlcNAc, idose, iduronic acid, glucuronic acid, sialicacid, polysaccharides related to the Thompsen-Friedrich antigens (Tn),and other monosaccharide or a disaccharide described herein.

In one particular embodiment, the peptide is a peptide of SEQ ID NO:2with 0-5, typically 0-3, often 1-3, more often 1 or 2 and most often 1additional amino acid residue(s).

Another aspect of the invention provides a method for treating aneurodegenerative disease in a subject. The method comprisesadministering to the subject in need of such a treatment atherapeutically effective amount of a glycopeptide of the presentinvention. The glycopeptide of the invention has a higher blood-brainbarrier penetration compared to the same peptide in the absence of asaccharide.

In other embodiments, the glycopeptide of the invention has a higherstability compared to the same peptide in the absence of the saccharide.

Still in other embodiments, neurodegenerative disease is selected fromthe group consisting of amyotrophic lateral sclerosis, Parkinson'sDisease, migraine attacks, traumatic brain injury, stroke, and dementia.

BRIEF DESCRIPTION OF THE DRAWINGS

FIG. 1 is a graph showing stability of PACAP₁₋₂₇, PACAP_(1-27-S-G) andPACAP_(1-27-S-L);

FIG. 2 is a bar graph showing half-life of PACAP₁₋₂₇ and itscorresponding serine glucosides in mouse serum;

FIG. 3 shows PAC₁-CHO calcium flux of PACAP₁₋₂₇ and various truncatedderivatives;

FIGS. 4A-4C (counter clock-wise from top left, respectively) are graphsshowing PACAP₁₋₂₇ and glycopeptides of the invention stimulation atvarious concentrations; and

FIGS. 5A-5D show PC12 cell morphology after vehicle versus PACAPtreatment at various solutions.

DETAILED DESCRIPTION OF THE INVENTION

As used in this specification and the appended claims, the singularforms “a,” “an” and “the” include plural references unless the contentclearly dictates otherwise.

The terms “saccharide” and “carbohydrate” are used interchangeablyherein and refer to aldoses and ketoses consisting of carbon (C),hydrogen (H) and oxygen (O) atoms, typically, but not necessarily, witha hydrogen-oxygen atom ratio of 2:1. The term also includesmono-deoxy-carbohydrates, such as deoxyribose, etc. where one hydroxygroup is removed from the empirical formula C_(m)(H₂O)_(n) formula,where m is typically 6 and n can be 5 or 6.

The terms “sugar” refers to a mono- and/or disaccharide.

The term “monosaccharide” refers to any type of hexose of the formulaC₆H₁₂O₆ or a derivative thereof. The ring structure (i.e., ring type) ofthe monosaccharide can be a pyranose or a furanose. In addition, themonosaccharides can be an α- or β-anomer. Monosaccharide can be aketonic monosaccharide (i.e., ketose), an aldehyde monosaccharide (i.e.,aldose), or any type of hexose of the formula C₆H₁₂O₆ or a derivativethereof. Exemplary aldoses of the invention include, but are not limitedto, allose, altrose, glucose, mannose, gulose, idose, galactose, talose,ribose, arabinose, xylose, lyxose, and derivatives thereof. Exemplaryketoses of the invention include, but are not limited to, psicose,fructose, sorbose, tagatose, ribulose, xylulose, and derivativesthereof. As used herein the term “derivative” refers to a derivative ofa monosaccharide in which one or more of the hydroxyl groups is replacedwith hydrogen (e.g., 2-deoxy glucose, 5-deoxyglucose, etc.), an amine(e.g., amino sugars) or is replaced with a halogen, such as chloro,fluoro or iodo, (e.g., 5-fluoroglucose, 2-fluoroglucose,5-chrologlucose, 2-chloroglucose, etc.). Monosaccharide can be an(L)-isomer or a (D)-isomer.

The term “disaccharide” refers to a carbohydrate composed of twomonosaccharides. It is formed when two monosaccharides are covalentlylinked to form a dimer. The linkage can be a (1→4) bond, a (1→6) bond, a(1→2) bond, etc. between the two monosaccharides. In addition, each ofthe monosaccharides can be independently an α- or β-anomer. Exemplarydisaccharides that can be used in the present invention include, but arenot limited to, sucrose, lactose, altose, maltose, trehalose,cellobiose, lactulose, and chitobiose, etc. Each of the monosaccharidescan independently be a ketonic monosaccharide (i.e., ketose), analdehyde monosaccharide (i.e., aldose), or any type of hexose of theformula C₆H₁₂O₆ or a derivative thereof. Exemplary aldoses that can beused in preparing disaccharides of the invention include, but are notlimited to, allose, altrose, glucose, mannose, gulose, idose, galactose,talose, ribose, arabinose, xylose, lyxose, and derivatives thereof.Exemplary ketoses that can be used in preparing disaccharides of theinvention include, but are not limited to, psicose, fructose, sorbose,tagatose, ribulose, xylulose, and derivatives thereof. Eachmonosaccharide can also be independently an (L)-isomer or a (D)-isomer.

“Treating” or “treatment” of a disease includes: (1) preventing thedisease, i.e., causing the clinical symptoms of the disease not todevelop in a mammal that may be exposed to or predisposed to the diseasebut does not yet experience or display symptoms of the disease; (2)inhibiting the disease, i.e., arresting or reducing the development ofthe disease or its clinical symptoms; or (3) relieving the disease,i.e., causing regression of the disease or its clinical symptoms.

As used herein, the term “treating”, “contacting” or “reacting” whenreferring to a synthesis or chemical reaction means adding or mixing twoor more reagents under appropriate conditions to produce the indicatedand/or the desired product. It should be appreciated that the reactionwhich produces the indicated and/or the desired product may notnecessarily result directly from the combination of two reagents whichwere initially added, i.e., there may be one or more intermediates whichare produced in the mixture which ultimately leads to the formation ofthe indicated and/or the desired product.

The terms “identical,” “identity,” “percent identity,” “percent sequenceidentity,” and “sequence identity” are used interchangeably herein. Inparticular, in the context of comparison of two or more peptides, theseterms refer to two or more sequences or subsequences that are the sameor have a specified percentage of nucleotides or amino acid residuesthat are the same, when compared and aligned for maximum correspondence,as measured using a sequence comparison algorithm known to one skilledin the art, or by visual inspection. For example, 75% sequence identityof a peptide A compared to peptide B means, 75% of the amino acidsequences in peptide A are the same as that of the amino acid sequencesof peptide B. The term also includes insertion/addition or deletion ofamino acids compared to a reference peptide. Thus, 75% sequence identityof peptide A compared to peptide B can also mean that peptide A has 25%more or 25% less (i.e., ±25%) amount of amino acid residues. Inparticular, if peptide B has 27 amino acids, then 75% sequence identifyof peptide A means peptide A can have from 21 to about 33 amino acidresidues. In some embodiments, these terms are used to denote sequenceswhich when aligned have similar (identical or conservatively replaced)amino acids in like positions or regions, where identical orconservatively replaced amino acids are those which do not alter theactivity or function of the protein as compared to the starting protein.Percent sequence identity may be calculated by determining the number ofresidues that differ between a peptide encompassed by the presentinvention and a reference peptide such as SEQ ID NOS: 1, 2 or 3), takingthat number and dividing it by the number of amino acids in thereference peptide (e.g., 27 or 27 amino acids), multiplying the resultby 100, and subtracting that resulting number from 100. For example, asequence having 35 amino acids with four amino acids that are differentfrom VIP would have a percent (%) sequence identity of 89% (e.g.100−((4/35)×100)). For a peptide having a sequence that is longer thanthe number of amino acids in a reference peptide, the number of residuesthat differ from the reference peptide will include the additional (ordifference in) amino acids over (or under) 35 for purposes of theaforementioned calculation. For example, a sequence having 37 aminoacids, with four amino acids different from the 35 amino acids in thereference peptide sequence and with two additional amino acids at thecarboxy terminus which are not present in the reference peptidesequence, would have a total of six amino acids that differ from thereference peptide. Thus, this sequence would have a percent (%) sequenceidentity of 83% (e.g. 100−((6/35)×100)). The degree of sequence identitymay be determined using methods well known in the art (sec, for example,Wilbur, W. J. et al., Proc. Natl. Acad. Science USA, 1983, 80, 726-730and Myers E. et al., Comput. Appl. Biosci., 1988, 4, 11-17. One programwhich may be used in determining the degree of similarity is theMegAlign Lipman-Pearson one pair method (using default parameters) whichcan be obtained from DNAstar Inc, 1128, Selfpark Street, Madison, Wis.,53715, USA as part of the Lasergene system. Another program, which maybe used, is Clustal W. This is a multiple sequence alignment packagedeveloped by Thompson et al. (Nucleic Acids Research, 1994, 22(22),4673-4680) for DNA or protein sequences. Clustal W is a general purposemultiple sequence alignment program for DNA or proteins. It producesbiologically meaningful multiple sequence alignments of divergentsequences. It calculates the best match for the selected sequences, andlines them up so that the identities, similarities and differences canbe seen.

“A therapeutically effective amount” means the amount of a compoundthat, when administered to a mammal for treating a disease, issufficient to effect such treatment for the disease. The“therapeutically effective amount” will vary depending on the compound,the disease and its severity and the age, weight, etc., of the mammal tobe treated.

The term “about” as used herein is not intended to limit the scope ofthe invention but instead encompass the specified material, parameter orstep as well as those that do not materially affect the basic and novelcharacteristics of the invention. The term “about” or “approximately” asused herein refers to being within an acceptable error range for theparticular value as determined by one of ordinary skill in the art,which will depend in part on how the value is measured or determined,i.e. the limitations of the measurement system, i.e. the degree ofprecision required for a particular purpose, such as a pharmaceuticalformulation. For example, “about” can mean within 1 or more than 1standard deviation, per the practice in the art. Alternatively, “about”can mean a range of ±20%, typically ±10%, often ±5% and more often ±1%of a given numeric value.

The present invention provides glycopeptides that are stable and cancross the blood-brain barrier. Glycopeptides are peptides that containcarbohydrate moieties (glycans or saccharides) covalently attached tothe side chains of the amino acid residues that constitute the peptide.In particular, glycopeptides of the invention include a peptide that iscovalently linked to a saccharide. The peptide portion of theglycopeptide of the invention has from about 20 to about 40 amino acidresidues and at least 75% sequence identity to pleiotropic peptidepituitary adenylate cyclase-activating polypeptide (PACAP) of SEQ IDNO:1 or vasoactive intestinal peptide (VIP) of SEQ ID NO:3. Thesaccharide portion of the glycopeptide of the invention ranges from 1 toabout 8 carbohydrates.

PACAP is a neuropeptide consisting of 38 amino acids. Two forms of thePACAP are known: PACAP-38 (i.e., PACAP₁₋₃₈) (SEQ ID NO:1) and PACAP-27(i.e., PACAP₁₋₂₇) (SEQ ID NO:2):

(SEQ ID NO: 1) 1       10         20         30       38HSDGIFTDSY SRYRKQMAVK KYLAAVLGKR YKQRVKNK (SEQ ID NO: 2)1       10         20      27 HSDGIFTDSY SRYRKQMAVK KYLAAVLAs the name indicates, PACAP-38 is a full-length PACAP having 38 aminoacid residue (i.e., SEQ ID NO:1) and PACAP-27 (i.e., amino acid residuesone to twenty-seven of SEQ ID NO:1) is a shorter version of PACAP.Interestingly, PACAP-27 has a 68% homology to VIP (SEQ ID NO:3), whichhas 28 amino acid residues:

(SEQ ID NO: 3) 1       10         20       28HSDAVFTDNY TRLRKQMAVK YYLNSILNPACAP was first isolated from bovine hypothalamus, and is known toregulate the development, maintenance, function, and plasticity of thenervous system, providing neuroprotective and neurotrophic support.PACAP has been shown to activate 3 closely-related G protein coupledreceptors: (i) PAC,, which has much higher affinity for PACAP, (ii)VPAC,, and (iii) VPAC₂ which bind both PACAP and VIP. They are expressedon neurons, microglia, and also by many other cell types. Without beingbound by any theory, it is believed constitutive expression of PACAP andits receptor PAC₁ confers neuroprotection to central visceromotorneurons via the PAC₁ receptor. PACAP also promotes cytodestructivefunctions of microglia (M1 amoeboid→M2 hypertrophic phenotype), thoughtto drive ALS disease progression via the VPAC₁ receptor. Thus, the idealdrugs for neuroprotection would be PAC, agonists at motor neurons topromote neuroprotection in case of ALS, or dopaminergic neurons in caseof PD, or hippocampal neurons in case of AD, and in each case VPAC₁antagonists at microglia to reduce inflammation by maintaining the M1(alternatively activated'/resolving anti-inflammatory cells) phenotypevs. the M2 (the classical, proinflammatory macrophages) microgliaphenotype or Tau-opathies.

The present invention is based at least in part in providing treatmentsfor ALS, PD, AD, and/or HD. In one aspect of the invention, aglycopeptide that can be used to treat a neurodegenerative disease. Inparticular, the glycopeptide of the invention includes a peptide that iscovalently linked to a saccharide. The peptide portion of theglycopeptides of the present invention has from about 20 to about 40amino acid residues and at least 75% sequence identity to pleiotropicpeptide pituitary adenylate cyclase-activating polypeptide (PACAP) ofSEQ ID NOs:1 or 2; or vasoactive intestinal peptide (VIP) of SEQ IDNO:3. The saccharide portion of the glycopeptides of the inventioninclude from 1 to about 8 carbohydrates.

In one embodiment, the glycopeptide is selected from the groupconsisting of PACAP₁₋₂₇, PACAP₁₋₃₈, VIP, [AC-His¹]PACAP-27,[Ala²]PACAP-27, [Gly²⁰]PACAP-27, and Ac-[Phe(pI)⁶, Nle¹⁷]-PACAP₁₋₂₇. Itshould be appreciated, the terms [Ac-His¹] PACAP-27, [Ala²]PACAP-27,[Gly²⁰]PACAP-27, and Ac-[Phe(pI)⁶, Nle¹⁷]-PACAP₁₋₂₇ have theconventional meaning well known to one skilled in the art. For example,[Ac-His¹] PACAP-27 means the histidine residue 1 in PACAP-27 isacetylated; [Ala²]PACAP-27 means the second amino acid residue ofPACAP-27, which is serine, is replaced by alanine; similarly[Gly²⁰]PACAP-27 means the 20^(th) amino acid residue of PACAP-27,namely, lysine, is replaced with glycine; and Ac-[Phe(pI)⁶,Nle¹⁷]-PACAP₁₋₂₇ means amino acid residues 6 and 17 of PACAP-27, namely,Phenylalanine and methionine, respectfully, are replaced with para-iodophenylalanine and norleucine (“Nle”), respectively.

In some embodiments, the glycopeptide of the invention is a retromodified peptide. The term “retro modified” refers to a peptide which ismade up of L-amino acids in which the amino acid residues are assembledin opposite direction to the native peptide with respect the which it isretro modified. In other embodiments, the glycopeptide of the inventionis an inverso modified peptide. The term “inverso modified” refers to apeptide which is made up of D-amino acids in which the amino acidresidues are assembled in the same direction as the native peptide withrespect to which it is inverso modified. Still in other embodiments, theglycopeptide of the invention is a retro-inverso modified peptide. Theterm “retro-inverso modified” refers to a peptide which is made up ofD-amino acids in which the amino acid residues are assembled in theopposite direction to the native peptide with respect to which it isretro-inverso modified. Thus, for example, if a normal (i.e., native)Link-N peptide (L-amino acids, N→C direction) is HSDGIFTDSY, thenRetro-inverso Link-N peptide (D-amino acids, C→N direction) is:HSDGIFTDSY. Retro peptide (L-amino acids, C→N direction) is: HSDGIFTDSY.And inverso peptide (D-amino acids, N→C direction) is: HSDGIFTDSY. Thesemodifications allow inter alio greater stability of peptides fromenzymatic and/or hydrolytic cleavage in vivo. In some embodiments, oneor more, typically one to ten, often one to five, and more often one tothree amino acid residues of the glycopeptide of the invention can be aD-isomer. In one particular embodiment, a single amino acid reside isreplaced with a D-isomer.

In one particular embodiment, glycopeptide [Ala²]PACAP-27 includes aD-isomer alanine, i.e., [D-Ala²]PACAP-27.

Still in another embodiment, at least one of the amino acid residues ofSEQ ID NO:1, 2 or 3 is substituted with a substitution amino acidresidue having a side-chain functional group that is glycosylated.Typically, the substitution amino acid residue comprises serine,threonine, hydroxyproline or a similar ethanolamine linker. As usedherein, the term “substitution amino acid” refers an amino acid residuethat is used to replace another amino acid in the same position. Incontrast, the term “additional amino acid residue” refers to an aminoacid residue that is added (i) in between two amino acid residues, (ii)at the C-terminus end of, or (iii) N-terminus end of SEQ ID NOs:1, 2 or3. In a similar manner, the term “deletion” or “deleted” amino acidresidue refers to an amino acid residue that is deleted or removed (i)from between two amino acid residues, (ii) at the C-terminus end of, or(iii) N-terminus end of SEQ ID NOs:1, 2 or 3.

In some embodiments, the glycopeptide has from about 25 to about 40,typically from about 25 to about 35, often about 25 to about 30, andmore often 27 to 29 amino acid residues. In one particular embodiment,the glycopeptide has from about 25 5o 30 amino acid residue and at least75%, typically at least 80%, often at least 85%, and more often at least90% sequence identity to SEQ ID NO:2.

Yet in other embodiments, the glycopeptide has at least 80%, typically85%, and often 90% sequence identity to PAPAC_(i-27) (SEQ ID NO:2).

Still yet in another embodiment, the glycopeptide of the invention hasat least 75%, typically at least 80%, often at least 85%, and more oftenat least 90% sequence identity to SEQ ID NO:2 and includes at least one,typically one to five, often one to three, and more often one additionalamino acid residue. In one particular embodiment, glycopeptides of theinvention has a 28 amino acid residue and at least 75%, typically atleast 80%, often at least 85%, and more often at least 90% sequenceidentity to SEQ ID NO:2.

Still in other embodiments, the glycopeptide of the present inventionhas at least one, typically, one to ten, often one to five, and moreoften one to five additional amino acid residues of SEQ ID NOs:1, 2, or3.

In yet other embodiments, the C-terminus end of the peptide isglycosylated.

Still yet in other embodiments, the glycopeptide is a pituitaryadenylate cyclase-activating polypeptide type I receptor (PAC₁) agonist.In other embodiments, the glycopeptide is a VPAC₁ agonist. Yet in otherembodiments, the glycopeptide is a selective PAC₁ and VPAC₁ agonist.Still in other embodiments, the glycopeptides of the invention are PAC₁agonist and VPAC₁ antagonist.

Yet in other embodiments, the PAC1 binding affinity (Ki) of theglycopeptide is less than about 50 nM, typically less than about 25 nM,and often less than about 10 nM.

In other embodiments, the VPAC1 binding affinity (Ki) of theglycopeptide is less than about 50 nM, typically less than about 25 nM,and often less than about 10 nM.

Still in other embodiments, the PAC1 agonist activity (Ki) of theglycopeptide is less than about 50 nM, typically less than about 25 nM,and often less than about 10 nM.

Yet in other embodiments, the VPAC1 agonist activity (κi) of saidglycopeptide is less than about 50 nM, typically less than about 25 nM,and often less than about 10 nM.

Agonistic and/or antagonistic activity of glycopeptides of the inventioncan be readily determined by one skilled in the art having read thepresent disclosure. For example, methods described by Doan et al., inBiochemical Pharmacology, 2011, 81, pp. 552-561, as well as in theExamples section below.

Typically, the saccharide comprises from 1 to 5, often 1 to 3carbohydrates. In one particular embodiment, the saccharide is amonosaccharide or a disaccharide.

Still yet in other embodiments, the peptide comprises a plurality ofglycosylated amino acid residues, i.e., different amino acid residuesare glycosylated where each saccharide linked to the amino acid residueis independently selected from those described herein. In someembodiments, 1 to 5, typically 1 to 3, often 1 or 2 different amino acidresidues are glycosylated. Often one amino acid residue is glycosylated.

In some embodiments, the saccharide is selected from the groupconsisting of with glucose, maltose, lactose, melibiose, maltotriose,sucrose, trehalose, altose, saccharose, maltose, cellobiose, gentibiose,isomaltose, primeveose, galactose, xylose, mannose, manosaminic acid,fucose, GalNAc, GlcNAc, idose, iduronic acid, glucuronic acid, sialicacid, polysaccharides related to the Thompsen-Friedrich antigens (Tn),and other monosaccharide and a disaccharide described herein.

In one particular embodiment, the glycopeptide of the invention includesa peptide portion having at least 75% sequence identity to SEQ ID NO:2and with 0-5, typically 0-3, often 1-3, more often 1 or 2 and most often1 additional amino acid residue(s).

Still in other embodiments, the glycopeptide of the invention has apeptide portion that is 27 or 28 amino acid residues in length and hasabout six or less, typically about five or less, and often four or lessamino acid residue difference compared to SEQ ID NO:2. Yet in otherembodiments, the glycopeptide of the invention has a peptide portionhaving one additional amino acid residue compared to SEQ ID NO:2. Insome embodiments, the additional amino acid residue is attached to theC-terminal end of SEQ ID NO:2. Yet in other embodiments, the additionalamino acid residue is glycosylated, i.e., covalently linked to asaccharide.

In other embodiments, glycopeptide of the invention has at least 75%,typically at least 80%, often at least 85%, more often at least 90%, andmost often at least 95% sequence identity to SEQ ID NO:4:

(SEQ ID NO: 4) 1       10         20      27 28HSDXXFXDSY SRYRKQXAVK KYLAAXX XAs can be seen, SEQ ID NO:4 is similar to SEQ ID NO:2 with oneadditional amino acid residue at the c-terminal end. Still in otherembodiments, glycopeptide of the invention is SEQ ID NO:4.

Methods for preparing glycopeptides of the invention can be readilyapparent to one skilled in the art having read the present disclosure.One method of preparing glycopeptides of the invention is usingFmoc-based solid-phase peptide synthesis. Briefly, in this methodinvolves covalently attaching a glycosyl group to the amino acidsequence by an O-linkage to a side chain in the address segment of thesequence. See, for example, as described by the present inventor inTetrahedron Asymmetry, 2005, 16, pp. 65-75, and the commonly assignedU.S. Pat. No. 5,767,254, issued to the present inventor, Robin Polt, allof which are incorporated herein by reference in their entirety. Anamino acid residue having a desired saccharide can be prepared using theprocedure described in the above cited U.S. Pat. No. 5,767,254. Thepeptide portion was synthesized using a solid state synthesis procedureas described in Tetrahedron Asymmetry, 2005, 16, pp. 66-75. Byadding/linking each desired amino acid to the solid phase synthesis andusing a desired glycosylated amino acid at an appropriate step of thesynthetic process, one can produce a wide range of glycopeptides of theinvention such as those exemplified in Table 1.

Yet in another embodiment, one or more serine amino acid residue of SEQID NO:2 or 4 is glycosylated. In one particular embodiment, thesaccharide moiety within this embodiment is independently selected fromthe group consisting of glucose, galactose, melibiose, xylose, lactose,trehalose, altose, or other monosaccharide or a disaccharide describedherein. Yet in another particular embodiment, one or more amino acid,e.g., serine, of SEQ ID NO:2 or 4 can be independently either L- orD-isomer.

Still in another embodiment, the glycopeptide is selected from thoselisted in Table 1 with a proviso that at least one amino acid residue isglycosylated. As can be seen, all glycopeptides in Table 1 fall withinthe scope of SEQ ID NO:4.

TABLE 1 Representative glycopeptides of SEQ ID NO: 4SEQ ID NO: 4 Representative Amino Acid Sequences H S D G I F T D  S ¹ Y  S ¹  R Y R K Q L A V K K Y L A A V L 

H s D G I F T D  S ¹  Y  S ¹  R Y R K Q 

 A V K K Y L A A V L  S* AcH   s  D G I F T D  S ¹  Y  S ¹  R Y R K Q 

 A V K K Y L A A V L  S* H  s  D G I F T D  S ¹  Y  S ¹  R Y R K Q 

 A V K K Y L A A V  S*  H  s  D G I F T D  S ¹  Y  S ¹  R Y R K Q 

 A V K K Y L A A  L   S*  L H  s  D G I F  A  D S Y S R Y R K Q 

 A V K K Y L A A V L  S* H  s  D G I F  A  D S Y S R Y R K Q 

 A V K K Y L A A V  S* H  s  D G I F  A  D S Y S R Y R K Q 

 A V K K Y L A A V  S* L H  s  D  a  I F T D S Y S R Y R K Q 

 A V K K Y L A A V L  S* H  s  D  Sar  IF T D S Y S R Y R K Q 

 A V K K Y L A A V L  S* H  s  

 I F T D S Y S R Y R K Q 

 A V K K Y L A A V L  S* H  s  D  Dava  I F T D S Y S R Y R K Q 

 A V K K Y L A A V L  S* H  s  D  A  I F T D S Y S R Y R K Q 

 A V K K Y L A A V L  S* H  s  D  B  I F T D S Y S R Y R K Q 

 A V K K Y L A A V L  S* H  s  D G  V  F T D S Y S R Y R K Q 

 A V K K Y L A A V L  S* H  s  D G  L  F T D S Y S R Y R K Q 

 A V K K Y L A A V L  S* H  s  D G  A  F T D S Y S R Y R K Q 

 A V K K Y L A A V L  S* H  s  D G  tL  F T D S Y S R Y R K Q 

 A V K K Y L A A V L  S* H  s  D G 

 F T D S Y S R Y R K Q 

 A V K K Y L A A V L  S* H  s  D G 

 F T D S Y S R Y R K Q 

 A V K K Y L A A V L  S* H  s  D G  G  F T D S Y S R Y R K Q 

 A V K K Y L A A V L  S* H  s  D  Sar   G  F T D S Y S R Y R K Q 

 A V K K Y L A A V L  S* H  s  D G  Sar  F T D S Y S R Y R K Q 

 A V K K Y L A A V L  S* H  s  D  Sar   Sar  F T D S Y S R Y R K Q 

 A V K K Y L A A V L  S* H  s  D  Sar   A  F T D S Y S R Y R K Q 

 A V K K Y L A A V L  S* H  s  D  A   Sar  F T D S Y S R Y R K Q 

 A V K K Y L A A V L  S* H  s  D  Sar   a  F T D S Y S R Y R K Q 

 A V K K Y L A A V L  S* H  s  D  a   Sar  F T D S Y S R Y R K Q 

 A V K K Y L A A V L  S* H  s  D  A   Å  F T D S Y S R Y R K Q 

 A V K K Y L A A V L  S* H  s  D  a   Å  F T D S Y S R Y R K Q 

 A V K K Y L A A V L  S* H  s  D  A   Å  F T D S Y S R Y R K Q 

 A V K K Y L A A V L  S* H  s  D  a   Å′  F T D S Y S R Y R K Q 

 A V K K Y L A A V L  S* H S D G I F T D  S ¹  Y  S ¹ R Y R K Q M A V K K Y L A A V L 

H S D G I F T D  S ¹  Y  S ¹  R Y R K Q M A V K K Y L A A V L  S ¹   H S ¹  D G I F T D  S ¹  Y  S ¹  R Y R K Q  X ¹⁷  A V K K Y L A A V L  S ¹  H  s ¹  D G I F T D  s/S ¹  Y  S ¹  R Y R K Q M A V K K Y L A A V L  S¹   H  S ¹  D G I F T D  S ¹  Y  S ¹  R Y R K Q M A V K K Y L A A V L s/S ¹   H S D G I F T D  S ¹  Y  S ¹  R Y R K Q M A V K K Y L A A V 

H S D G I F T D  S ¹  Y  S ¹  R Y R K Q M A V K K Y L A A V  S ¹   H  S¹  D G I F T D  S ¹  Y  S ¹  R Y R K Q M A V K K Y L A A V  S ¹   H  s ¹ D G I F T D  s/S ¹  Y  S ¹  R Y R K Q M A V K K Y L A A V  S ¹   H  S ¹ D G I F T D  S ¹  Y  S ¹  R Y R K Q M A V K K Y L A A V  s/S ¹   Mel =melibiose; s = D-Serine;

 = L-Nor-Leucine; S* = L-Ser(β-D-Glc);

 = L-Ser(β-Melibiose); Sar = Sarcosine; a = D-Alanine; βA = β-Alanine;Dava = Diaminovaleric Acid; B = α-Aminoisobuteric Acid; tL =L-tert-Leucine; Nva = L-nor-Valine; Å = L-N-Methylalanine; Å′ =D-N-Methylalanine; X¹⁷ = M or Ñ at 17-position; ¹ = each is optionallyand independently glycosylated with glucose, galactose, melibiose,xylose, lactose, trehalose, altose, or other monosaccharide or adisaccharide described herein; and s/S = D- or L- isomer of serine,respectively. In all cases, at least one of the amino acid residue isglycosylated.

The ability of glycopeptides of the invention to cross the blood-brainbarrier can be readily determined by one skilled in the art having readthe present disclosure. Exemplary examples for determining theblood-brain barrier crossing can be found in the Examples section below.

In some embodiments, the in vivo plasma half-life (t_(1/2)) ofglycopeptides of the present invention is at least 20 min, typically atleast 30 min, often at least 40 min and more often at least 60 min.Alternatively, the half-life of glycopeptides of the invention is atleast about 600% or more, typically at least about 1200% or more, oftenat least about 1400% or more, and more often at least 1700% or more thanthe same peptide in the absence of the saccharide portion.

Still in other embodiments, the amount of blood-brain barrier crossingby glycopeptides of the invention is increased by at least about 1000%,typically at least about 1200%, often at least about 1500%, and moreoften at least about 1700% compared to the same peptide in the absenceof the saccharide portion.

Some representative agonist data for glycopeptides of SEQ ID NO:4 areprovided in Table 2 below.

PQC1 VPAC1 VPAC2 EC₅₀ E_(Max) EC₅₀ EC_(Max) EC₅₀ E_(Max) Sequence (nM)(%) (nM) (%) (nM) (%) HSDGIFTDSYSRYRKQMAVKKYLAAVL    0.4, 100 14.8 ± 1.6100 0.35 ± 0.16 100    0.13,    0.34 HSDGIFTDSYSRYRKQ L AVKKYLAAVL-Ser(Mel)    0.57  99 0.55 102 9.4  86 H s DGIFTDSYSRYRKQ

AVKKYLAAVL- Ser(Glc)   25.5  85 1.3  90 241 104 H s DGIFTDSYSRYRKQ

AVKKYLAAV- Ser(Glc)   54.5  86 4.8  90 654 107 H s DGIFTDSYSRYRKQ

AVKKYLAAL- Ser(Glc) -L >250 −79 5.9  93 >2500 −95 H s DGIF A DSYSRYRKQ

AVKKYLAAVL- Ser(Glc) >250 −71 78.8  93 >2500 −42 H s DGIFADSYSRYRKQ

AVKKYLAAV- Ser(Glc) NC NC 1366  85 NC NC H s DGIFADSYSRYRKQ

AVKKYLAAV- Ser(Glc) -L NC NC 1623  78 NC NC

Chemical stability of the glycopeptides in vivo clearly plays animportant role in the deliverability of the drugs to the site(s) ofaction within the brain. It is also important to know what the chemicalor metabolic instabilities are in order to inform the drug designprocess. To this end, stability of PACAP₁₋₂₇; PACAP_(1-27-S-G) andPACAP_(1-27-S-L) was tested. As shown in FIG. 1, PACAP₁₋₂₇ and itsglycosylated analogues degraded over 30 min in mouse serum at 37° C.Data were fitted using a single exponential decay model (R²>0.71, in allcases). FIG. 2 shows that Serine glucoside (PACAP_(1-27-S-G), Glc)showed a significant increase in mouse serum t_(1/2) in vitro comparedto the native peptide PACAP₁₋₂₇ and the corresponding lactoside(PACAP_(1-27-S-L), Lac) when compared using a 1-way analysis of variance(F2=12.91, p=0.0067, Tukey's multiple comparison Native vs Glc, q=5.760p<0.05, Lac vs Glc, q=6.602 p<0.5).

Using CHO cells that express human PAC₁ receptors, PACAP₁₋₂₇, theglucoside PACAP_(27-S-G), and the truncated putative antagonistPACAP₆₋₂₇ and its derivatives were tested as agonists using FLIPR. Itwas found that PACAP₁₋₂₇ and PACAP_(1-27-S-G), the serine glucoside,activated PAC₁ with high potency (0.95±0.4 nM and 5.68±2.3 nM,respectively). See FIG. 3. In addition, the normalized efficacy of thePACAP_(1-27-S-G) glucoside was nearly identical to the native PACAP₁₋₂₇peptide, at 101.9±1.6%. These findings strongly indicate thatglycosylation of PACAP₁₋₂₇ does not significantly alter binding andactivation of the PAC₁ receptor, supporting the use of such aglycopeptide for therapeutic purposes. As expected, none of thePACAP₆₋₂₇ derivatives showed agonist activity at concentrations up to 1μM. See FIG. 3.

PAC₁-CHO calcium flux activation was measured using FLIPR in response to11 point concentration curves of PACAP₁₋₂₇, the glucosidePACAP_(1-27-S-G), and the truncated derivatives (putative antagonists)of PACAP₆₋₂₇. Response was measured over 10 minutes, the max-mincalculated, and all data was normalized to the maximum response causedby PACAP₁₋₂₇ (100%) and vehicle (0%). The mean±SEM is shown, using themean value from each independent experiment. N=3 independent experimentsperformed, 3 variable non-linear curve fit using Prism. PACAP₁₋₂₇ andPACAP_(1-27-S-G) showed potent, efficacious agonist activity.

The ability of glycosylated and non-glycosylated PACAP₆₋₂₇ derivativesto block activation of the PAC₁ receptor by PACAP₁₋₂₇ was also tested. Avariable concentration mode antagonist assay versus 5 nM of PACAP₁₋₂₇ inthe PAC₁-CHO cells was employed using FLIPR. Surprisingly, nosignificant antagonist activity of PACAP₆₋₂₇ or any derivative at aconcentration up to 1μM was detected. See FIG. 4A-C. When the knownantagonist PACAP₆₋₃₈ was tested, only a low potency antagonism wasdetected (>333 nM). See FIG. 4A. These findings are at odds with amolecular pharmacology study of PACAP₆₋₃₈ and PACAP₆₋₂₇ with reportedK_(i) values of 1.5 and 60 nM, respectively.

The ability of PACAP₆₋₂₇ derivatives to block PACAP₁₋₂₇ induced calciumflux was measured using FLIPR. FIG. 4A shows variable concentration modeantagonist experiments. Concentration curves of PACAP₆₋₃₈, PACAP₆₋₂₇,and PACAP₆₋₂₇ derivatives were added to the cells for 2 minutes,followed by 5 nM of PACAP₁₋₂₇. The max-min response was determined, andnormalized to the stimulation caused by 5 nM PACAP₁₋₂₇ (100%) andvehicle (0%). Only PACAP₆₋₃₈ showed antagonism, but it is low potency.FIG. 4B shows fixed concentration mode experiments with PACAP₆₋₃₈. Fixedconcentrations of PACAP₆₋₃₈ was added to cells for 2 minutes, followedby concentration curves of PACAP₁₋₂₇. The max-min response wasdetermined, and normalized to the max response of the PACAP₁₋₂₇ curvewithout antagonist present (100%) and vehicle (0%). PACAP₆₋₃₈ shiftedthe curve only at the highest concentration (1 μM). FIG. 4C shows fixedconcentration mode experiments with PACAP₆₋₂₇, performed as in FIG. 4BN=3 independent experiments. PACAP₆₋₂₇ showed no detectable shifts inthe agonist curves.

Fixed concentration antagonist mode experiments was performed with thepeptides PACAP₆₋₂₇ and PACAP₆₋₃₈. It was found that PACAP₆₋₂₇ caused noshift in the agonist curves, while PACAP₆₋₃₈ induced a shift only at 1μM. See FIGS. 4B and 4C. This resulted in a pA2 value of 200.6±55.4 nMfor PACAP₆₋₃₈, well above the 1.5 nM value previously reported. Notably,PACAP₆₋₃₈ also showed a Schild Slope of 2.0±0.1. A Schild Slope of 1fits the assumptions of the model, while a slope above 1 suggests thatthe compound is more effective than would be expected for competitiveantagonism. Without being bound by any theory, it is believed that thisresult may be due to the short incubation times in the FLIPR assay,which might not be long enough to allow the system to reach equilibrium,thus leading to a relatively high Schild Slope. Alternatively, PACAP₆₋₃₈may function by a different mechanism, e.g., binding to VPAC_(1/2).

PC12 cells are non-adherent cells, and in spite of using thepoly-D-Lysine coated plates, the majority of the cells remainedsuspended. During the media exchange many of the cells were removed withthe spent media. The remaining cells could be visually evaluated forqualitative morphological changes at the end of the treatment period,but meaningful cell quantification could not be done reliably using thisapproach. It was found that glucoside and lactoside PACAP₁₋₂₇ derivativetreatment produced neurite outgrowth and arborization when compared tovehicle treated cells. FIGS. 5A-D. Qualitatively, it appeared that thearborization caused by PACAP₁₋₂₇ may be more extensive than that causedby the glucoside and lactoside derivatives, but again this could not bequantified. In any case, both PACAP₁₋₂₇ and the derivatives inducedneurite outgrowth, suggesting native PAC₁ agonist activity.

FIGS. 5A-5D show PC12 Cell Morphology after Vehicle vs PACAP Treatment(100 nM). FIG. 5A: diluent only; 5B: PACAP₁₋₂₇; 5C: PACAP_(1-27-S-G);5D: PACAP_(1-27-S-L). The cell body volumes all showed increases whentreated with each of the PACAP derivatives. In all cases the processoutgrowths on the treated cells were greater than 2× the cell body width

Endogenous PACAP peptides occur as C-terminal peptide amides that haveeither 27 (10%) or 38 (90%) amino acid residues, and are typicallyregarded as PAC₁ agonists in assays using intact tissue or in cellculture. For the present studies, a separate CHO cell line expressingthe PAC₁ receptor individually was developed. Use of solid-phase peptidesynthesis allowed a wide different glycopeptides as illustrated in Table1 above. As can be seen, glycopeptides of the invention retained theiragonist activity on PC12 cell cultures (FIGS. 5A-5D) and in thequantitative CHO cell assay (FIG. 3). In addition, glycopeptides of theinvention had a higher half-life in mouse serum (FIGS. 4A-4C) comparedto a corresponding non-glycosylated peptides. More significantly,experiments showed glycopeptides of the invention can cross theblood-brain barrier in mice (FIGS. 4A-4C). In use, an effective amountof glycopeptides of the present invention can be administered to apatient in need of treatment in a therapeutically effective unit dosedelivery amount of between about 0.1 and 10 milligrams per kilo,typically 1-2 doses per day, or even less frequently. The glycopeptidesof the invention may be delivered in a pharmaceutically acceptablecarrier.

Pharmaceutical formulations and pharmaceutical compositions are wellknown in the art, and can be found, for example, in Remington'sPharmaceutical Sciences, Mack Publishing Co., Easton, Pa., USA, which ishereby incorporated by reference in its entirety. Any formulationsdescribed therein or otherwise known in the art are embraced byembodiments of the disclosure.

Pharmaceutical excipients are well known in the art and include, but arenot limited to, saccharides such as, for example, lactose or sucrose,mannitol or sorbitol, cellulose preparations, calcium phosphates such astricalcium phosphate or calcium hydrogen phosphate, as well as binders,such as, starch paste such as, for example, maize starch, wheat starch,rice starch, potato starch, gelatin, tragacanth, methyl cellulose,hydroxypropylmethylcellulose, sodium carboxymethylcellulose, polyvinylpyrrolidone or combinations thereof.

In particular embodiments, pharmaceutical formulations may include theactive compound described and embodied above, a pharmaceuticallyacceptable carrier or excipient and any number of additional orauxiliary components known in the pharmaceutical arts such as, forexample, binders, fillers, disintegrating agents, sweeteners, wettingagents, colorants, sustained release agents, and the like, and incertain embodiments, the pharmaceutical composition may include one ormore secondary active agents. Disintegrating agents, such as starches asdescribed above, carboxymethyl-starch, cross-linked polyvinylpyrrolidone, agar, alginic acid or a salt thereof, such as sodiumalginate and combinations thereof. Auxiliary agents may include, forexample, flow-regulating agents and lubricants, such as silica, talc,stearic acid or salts thereof, such as magnesium stearate or calciumstearate, polyethylene glycol and combinations thereof. In certainembodiments, dragee cores may be prepared with suitable coatings thatare resistant to gastric juices, such as concentrated saccharidesolutions, which may contain, for example, gum arabic, talc, polyvinylpyrrolidone, polyethylene glycol, titanium dioxide, lacquer solutionsand suitable organic solvents or solvent mixtures and combinationsthereof. In order to produce coatings resistant to gastric juices,solutions of suitable cellulose preparations, such as acetylcellulosephthalate or hydroxypropylmethyl-cellulose phthalate may also be used.In still other embodiments, dye stuffs or pigments may be added to thetablets or dragee coatings, for example, for identification or in orderto characterize combinations of active compound doses.

Pharmaceutical compositions of the disclosure can be administered to anyanimal, and in particular, any mammal, that may experience a beneficialeffect as a result of being administered a compound of the disclosureincluding, but not limited to, humans, canines, felines, livestock,horses, cattle, sheep, and the like. The dosage or amount of at leastone compound according to the disclosure provided pharmaceuticalcompositions of embodiments may vary and may depend, for example, on theuse of the pharmaceutical composition, the mode of administration ordelivery of the pharmaceutical composition, the disease indication beingtreated, the age, health, weight, etc. of the recipient, concurrenttreatment, if any, frequency of treatment, and the nature of the effectdesired and so on. Various embodiments of the disclosure includepharmaceutical compositions that include one or more compounds of thedisclosure in an amount sufficient to treat or prevent diseases such as,for example, cancer. An effective amount of the one or more compoundsmay vary and may be, for example, from about 0.1 to 10 milligrams perkilo, typically 1-2 doses per day.

The pharmaceutical compositions of the disclosure can be administered byany means that achieve their intended purpose. For example, routes ofadministration encompassed by the disclosure include, but are notlimited to, subcutaneous, intravenous, intramuscular, intraperitoneal,buccal, or ocular routes, rectally, parenterally, intrasystemically,intravaginally, topically (as by powders, ointments, drops ortransdermal patch), oral or nasal spray are contemplated in combinationwith the above described compositions.

Embodiments of the disclosure also include methods for preparingpharmaceutical compositions as described above by, for example,conventional mixing, granulating, dragee-making, dissolving,lyophilizing processes and the like. For example, pharmaceuticalcompositions for oral use can be obtained by combining the one or moreactive compounds with one or more solid excipients and, optionally,grinding the mixture.

Suitable auxiliaries may then be added and the mixture may be processedto form granules which may be used to form tablets or dragee cores.Other pharmaceutical solid preparations include push-fit capsulescontaining granules of one or more compound of the disclosure that can,in some embodiments, be mixed, for example, with fillers, binders,lubricants, stearate, stabilizers or combinations thereof. Push-fitcapsules are well known and may be made of gelatin alone or gelatin incombination with one or more plasticizer such as glycerol or sorbitol toform a soft capsule. In embodiments in which soft capsules are utilized,compounds of the disclosure may be dissolved or suspended in one or moresuitable liquids, such as, fatty oils or liquid paraffin and, in somecases, one or more stabilizers.

Liquid dosage formulations suitable for oral administration are alsoencompassed by embodiments of the disclosure. Such embodiments, mayinclude one or more compounds of the disclosure in pharmaceuticallyacceptable emulsions, solutions, suspensions, syrups, and elixirs thatmay contain, for example, one or more inert diluents commonly used inthe art such as, but not limited to, water or other solvents,solubilizing agents and emulsifiers such as ethyl alcohol, isopropylalcohol, ethyl carbonate, ethyl acetate, benzyl alcohol, benzylbenzoate, propylene glycol, 1,3-butylene glycol, dimethyl formamide,oils (for example, cottonseed, groundnut, corn, germ, olive, castor, andsesame oils), glycerol, fatty acid derivatives of glycerol (for example,labrasol), tetrahydrofurfuryl alcohol, polyethylene glycols and fattyacid esters of sorbitan, and mixtures thereof. Suspensions may furthercontain suspending agents as, for example, ethoxylated isostearylalcohols, polyoxyethylene sorbitol and sorbitan esters, microcrystallinecellulose, aluminum metahydroxide, bentonite, agar-agar, and tragacanth,and mixtures thereof.

Formulations for parenteral administration may include one or morecompounds of the disclosure in water-soluble form, for example,water-soluble salts, alkaline solutions, and cyclodextrin inclusioncomplexes in a physiologically acceptable diluent which may beadministered by injection. Physiologically acceptable diluent of suchembodiments, may include, for example, sterile liquids such as water,saline, aqueous dextrose, other pharmaceutically acceptable sugarsolutions; alcohols such as ethanol, isopropanol or hexadecyl alcohol;glycols such as propylene glycol or polyethylene glycol; glycerol ketalssuch as 2,2-dimethyl-1,3-dioxolane-4-methanol; ethers such aspoly(ethyleneglycol)400; pharmaceutically acceptable oils such as fattyacid, fatty acid ester or glyceride, or an acetylated fatty acidglyceride. In some embodiments, formulations suitable for parenteraladministration may additionally include one or more pharmaceuticallyacceptable surfactants, such as a soap or detergent; suspending agentsuch as pectin, carbomers, methylcellulose,hydroxypropylmethylcellulose, or carboxymethylcellulose; an emulsifyingagent; pharmaceutically acceptable adjuvants or combinations thereof.Additional pharmaceutically acceptable oils which may be useful in suchformulations include those of petroleum, animal, vegetable or syntheticorigin including, but not limited to, peanut oil, soybean oil, sesameoil, cottonseed oil, olive oil, sunflower oil, petrolatum, and mineraloil; fatty acids such as oleic acid, stearic acid, and isostearic acid;and fatty acid esters such as ethyl oleate and isopropyl myristate.Additional suitable detergents include, for example, fatty acid alkalimetal, ammonium, and triethanolamine salts; cationic detergents such asdimethyl dialkyl ammonium halides, alkyl pyridinium halides, andalkylamine acetates; and anionic detergents, such as alkyl, aryl, andolefin sulfonates, alkyl, olefin, ether and monoglyceride sulfates, andsulfosuccinates. In some embodiments, non-ionic detergents including,but not limited to, fatty amine oxides, fatty acid alkanolamides andpolyoxyethylenepolypropylene copolymers or amphoteric detergents such asalkyl-β-aminopropionates and 2-alkylimidazoline quaternary salts, andmixtures thereof may be useful in parenteral formulations of thedisclosure.

Pharmaceutical compositions for parenteral administration may containfrom about 0.5 to about 25% by weight of one or more of the compounds ofthe disclosure and from about 0.05% to about 5% suspending agent in anisotonic medium. In various embodiments, the injectable solution shouldbe sterile and should be fluid to the extent that it can be easilyloaded into a syringe. In addition, injectable pharmaceuticalcompositions may be stable under the conditions of manufacture andstorage and may be preserved against the contaminating action ofmicroorganisms such as bacteria and fungi.

Topical administration includes administration to the skin or mucosa,including surfaces of the lung and eye. Compositions for topicaladministration, may be prepared as a dry powder which may be pressurizedor non-pressurized. In non-pressurized powder compositions, the activeingredients in admixture are prepared as a finely divided powder. Insuch embodiments, at least 95% by weight of the particles of theadmixture may have an effective particle size in the range of 0.01 to 10micrometers. In some embodiments, the finely divided admixture powdermay be additionally mixed with an inert carrier such as a sugar having alarger particle size, for example, of up to 100 micrometers in diameter.Alternatively, the composition may be pressurized using a compressedgas, such as nitrogen or a liquefied gas propellant. In embodiments, inwhich a liquefied propellant medium is used, the propellant may bechosen such that the compound and/or an admixture including the compounddo not dissolve in the propellant to any substantial extent. In someembodiments, a pressurized form of the composition may also contain asurface-active agent. The surface-active agent may be a liquid or solidnon-ionic surface-active agent or may be a solid anionic surface-activeagent, which in certain embodiments, may be in the form of a sodiumsalt.

Compositions for rectal administration may be prepared by mixing thecompounds or compositions of the disclosure with suitable non-irritatingexcipients or carriers such as for example, cocoa butter, polyethyleneglycol or a suppository wax. Such carriers may be solid at roomtemperature but liquid at body temperature and therefore melt in therectum and release the drugs.

In still other embodiments, the compounds or compositions of thedisclosure can be administered in the form of liposomes. Liposomes aregenerally derived from phospholipids or other lipid substances that formmono- or multi-lamellar hydrated liquid crystals when dispersed in anaqueous medium. Any non-toxic, physiologically acceptable andmetabolizable lipid capable of forming liposomes can be used, and inparticular embodiments, the lipids utilized may be natural and/orsynthetic phospholipids and phosphatidyl cholines (lecithins). Methodsto form liposomes are known in the art (see, for example, Prescott, Ed.,Meth. Cell Biol. 14:33 (1976), which is hereby incorporated by referencein its entirety). Compositions including one or more compounds of thedisclosure in liposome form can contain, for example, stabilizers,preservatives, excipients and the like.

In general, methods of embodiments of the disclosure may include thestep of administering or providing an “effective amount” or a“therapeutically effective amount” of a compound or composition of thedisclosure to an individual. In such embodiments, an effective amount ofthe compounds of the disclosure may be any amount that produces thedesired effect. As described above, this amount may vary depending on,for example, the circumstances under which the compound or compositionis administered (e.g., to incite treatment or prophylactically), thetype of individual, the size, health, etc. of the individual and so on.The dosage may further vary based on the severity of the condition. Forexample, a higher dose may be administered to treat an individual with awell-developed metastatic condition, compared to the amount used toprevent a subject from developing the metastatic condition. Thoseskilled in the art can discern the proper dosage based on such factors.For example, in some embodiments, the dosage may be within the range ofabout 0.01 mg/kg body weight to about 10 mg/kg body weight.

The administration schedule may also vary. For example, in someembodiments, the compounds or compositions of the disclosure may beadministered in a single dose once per day or once per week. In otherembodiments, the compounds or compositions of the disclosure may beadministered in one or two or more doses per day. For example, in oneembodiment, an effective amount for a single day may be divided intoseparate dosages that may contain the same or a different amount of thecompound or composition and may be administered several times throughouta single day. Without wishing to be bound by theory, the dosage peradministration and frequency of administration may depend, for example,on the specific compound or composition used, the condition beingtreated, the severity of the condition being treated, and the age,weight, and general physical condition of the individual to which thecompound or composition is administered and other medications which theindividual may be taking. In another exemplary embodiment, treatment maybe initiated with smaller dosages that are less than the optimum dose ofthe compound, and the dosage may be increased incrementally until a moreoptimum dosage is achieved.

In each of the embodiments above, the compound administered can beprovided as a pharmaceutical composition including compound as describedabove and a pharmaceutically acceptable excipient or a pure form of thecompound may be administered.

In additional embodiments, the compound or composition of the disclosuremay be used alone or in combination with one or more additional agents.For example, in some embodiments, a compound or composition ofdisclosure may be formulated with one or more additional anti-canceragents or combinations thereof such that the pharmaceutical compositionobtained including the compound or composition of the disclosure and theone or more additional agents can be delivered to an individual in asingle dose. In other embodiments, the compound or composition of thedisclosure may be formulated as a separate pharmaceutical compositionthat is delivered in a separate dose from pharmaceutical compositionsincluding the one or more additional agents. In such embodiments, two ormore pharmaceutical compositions may be administered to delivereffective amounts of a compound or composition of the disclosure and theone or more additional agents.

Additional objects, advantages, and novel features of this inventionwill become apparent to those skilled in the art upon examination of thefollowing examples thereof, which are not intended to be limiting. Inthe Examples, procedures that are constructively reduced to practice aredescribed in the present tense, and procedures that have been carriedout in the laboratory are set forth in the past tense.

EXAMPLES

Glycopeptides of the invention were prepared using one of the followingprocedures.

General Method I. The C-terminal amino acids were loaded onto Fmoc-Rinkresin (Advanced ChemTech, Louisville, Ky., USA) at 0.1 mmol/g resinloading in 25 mL fritted syringes and dimethylformamide (˜5 mL per gramresin) was added. The mixture was stirred at RT for two minutes (x2). Asolution of 2% DBU and 3% piperidine in DMF (v:v) was added and themixture was agitated for 5 minutes, refreshed, and agitated for anadditional 10 minutes. The resin was washed with DMF (x5), and finallywith N-methylpyrrolidine (NMP). In a separate vial, a solution ofFmoc-β-OGlc(OAc)₄-Ser-OH (0.12 mmol) and HOBt.H₂O (0.13 mmol) in 5 mLNMP was prepared. To this solution was added DIC (0.26 mmol) and themixture was stirred for 5 minutes. The resulting solution was added tothe resin and agitated for 10 minutes. Next, the syringe was placed in amicrowave oven (Emerson 900 W Microwave—MW9338SB) and irradiated atpower level 1 for 10 minutes. The syringe was then agitated at RT for anadditional 30 minutes. The resin was washed with NMP (x1), DMF (x5), andCH₂Cl₂ (x5), and dried in vacuo overnight.

General Method II: Glycopeptides were assembled on a Prelude® PeptideSynthesizer (Protein Technologies, Inc., Tucson, Ariz., USA) using thefollowing procedure.

Rink resin (100 mg) was placed into the fritted reaction vessels. Aminoacids were dissolved in DMF at 250 mM concentration, HATU at 375 mM, andTMP at 3M. The following steps were performed for coupling: DMF Top Wash(1.5 mL, 2 min mix and drain; x6), Deprotection (2% DBU/3% piperidine inDMF; 1.5 mL, 4 min mix and drain; 8 min mix and drain), DMF Top Wash(1.5 ml, 2 min mix and drain; x5), Amino Acid Building Block (0.950 mL,30 sec mix), Activator 1 (HATU, 0.650 mL, 30 sec mix), Base (TMP, 0.300mL, 35 min mix and drain), DMF Top Wash (1.5 mL, 2 min mix and drain;x2). After coupling aspartic acid D7, the deprotection solution waschanged to 0.1 M HOBt.H₂O/5% piperazine in DMF to minimize aspartimideformation.

Cleavage of the glycopeptides from the resin. The glycopeptides preparedusing General Method I or II were cleaved from the resin using a mixtureof trifluoroacetic acid, triethylsilane, water, methylene chloride andmethoxyphenyl (9:0.3:0.2:1:0.05 by volume). Briefly, the mixture wasagitated at room temperature (RT) for 2 hours. The resulting solutionswere transferred to 15 mL centrifuge tubes, evaporated under argon,precipitated in ice-cold Et₂O, decanted, and rewashed with Et₂O, thendissolved in H₂O and lyophilized to afford the crude material as fluffywhite solids.

Purification of the crude glycopeptides was accomplished by ReversedPhase HPLC (RP-HPLC) with a preparative RP (C-18) Phenomenex (250×22 mm)column using a CH₃CN—H₂O gradient solvent system containing 0.1%trifluoroacetic acid. Homogeneity of the purified glycopeptides wasconfirmed by analytical RP-HPLC and high resolution mass spectrometry.

Binding Assay.

Binding assay to determine PAC1, VPAC1, and VPAC2 agonist activity canbe performed as described by Doan et al. in Biochem. Pharmalcol., 2011,81, pp. 552-561. Briefly, the binding assay procedure is as follows:Acetylated PACAP27 was radio-iodinated using the chloramine-T technique(Hunter WMGF, Nature, 1962,194, pp. 495-6) and purified on a Sep-Pak C18cartridge (Waters, Milford, Mass., USA). CHO cells expressing one of thePACAP-related receptors were seeded at a density of 125,000 cells perwell in 24-well plates. After 24 h, the culture medium was removed, andcells were first incubated at room temperature for 10 min in bindingbuffer (0.1% BSA, 25 mM Tris-HCl, 25 mM MgCl₂, and 5mg/L bacitracin, pH7.4) and then exposed to increasing peptide concentrations in thepresence of 0.05 nM ¹²⁵I-Ac-PACAP27. After 2 h at room temperature,cells were washed twice with binding buffer, lysed (0.1 M NaOH), and thecell-bound radioactivity was quantified using a g-counter (1470Automatic Gamma Counter, Perkin Elmer). Results were expressed as thepercentage of the specific binding of ¹²⁵I-Ac-PACAP27 obtained in theabsence of competitive ligands. Nonspecific binding was determined inthe presence of 10 mM PACAP38 and averaged at 5-10% of total binding.

Biological Assay.

The neuroprotective effects of the PAC₁ agonist and theanti-inflammatory effects of the VPAC₁ antagonist in cell culture modelswere tested. In addition, stability of peptides and BBB penetration invivo were also tested. These tests were used to identify peptides fortherapeutic candidates in vivo in preclinical models of ALS and PD.

The overall process was as follows: Flow-injection tandem massspectrometry (FI-MS^(II)) was used to observe the degradation of thepeptides and glycopeptides with a Thermo LCQ with electrosprayionization (ESI). The technique involved injection of a sample bolus ofmaterial in mouse serum via a six port valve with fluid flow deliveredvia a syringe pump, and subsequent electrospray ionization (ESI)followed by mass spectral analysis. Samples were diluted to aconcentration of ˜5 μM of each PACAP analogue, and were incubated at 37°C. for times varying from 1 to 60 minutes. After samples had beenincubated for the prescribed amount of time they were prepared for massspectrometry analysis by withdrawing 10 microliters of solution andspiking with 1 microliter of a 10 μM solution of peptide internalstandard (angiotensin II) in 50% acetic acid and subjecting them to astandard C18 zip tip desalting. These solutions, once eluted from thezip tip were diluted to 100 μL in 50:50 acetonitrile/water with 0.1%formic acid. Tandem mass spectrometry analysis (MS³) was conducted toyield specific, quantitative signals proportional to the amount of PACAPanalogue at each time point. This technique was also used withmicrodiasylate samples from a mouse after i.p. administration ofPACAP1-27-S-G.

A custom DNA clone of the human PAC1 gene with 3 hemagglutinin (HA) tagsinserted 3′ to the signal peptide sequence (to avoid proteolytic loss)was obtained from Genecopoeia (Rockville, Md.). The construct waselectroporated into Chinese Hamster Ovary (CHO) cells, and selected forwith 500 μg/mL of G418. The resulting population was screened for highexpressing clones, and one such clone selected for further analysis. Theclonal cell line (PAC₁-CHO) displayed high receptor expression byimmunocytochemistry and Western blot, and showed selective activation ofsignaling in response to PACAP₁₋₂₇. This cell line was used for allmolecular pharmacology experiments. The cells were maintained inDMEM/F12 with 10% heat-inactivated FBS, 1× penicillin/streptomycin, and500 μg/mL G418, at 37° C. and 5% CO₂.

All molecular pharmacology experiments were carried out using a FLIPRTetra from Molecular Devices (Sunnyvale, Calif.), set to image calciumflux using the manufacturer's recommended settings and protocols. Theday before an experiment, the PAC₁-CHO cells were split into 384 wellblack walled, clear bottom microplates, 10,000 cells per well. The cellswere recovered overnight in growth medium (as above). The next day, thegrowth medium was replaced with Calcium 6 dye (Molecular Devices) usingthe manufacturer recommended buffer with 2.5 mM probenecid. The cellswere incubated for 2 hours in the culture incubator, and removed duringthe last 15 minutes to allow equilibration to room temperature. Compoundas indicated below was added to the cells using a 384 tip block, withreal time monitoring before, during, and 15 minutes after compoundaddition. The resulting calcium flux was recorded, and themaximum-minimum response over the entire observation time calculated andreported as the mean±SEM (4 wells per point).

For agonist mode experiments, compound was added in an 11 pointconcentration curve, with a vehicle control (buffer). The resultingresponse was normalized to the stimulation caused by PACAP₁₋₂₇ (100%)and vehicle (0%). The response was analyzed using a 3 variablenon-linear curve fit, and the EC₅₀ (nM) and E_(Max) (%) calculated andreported (Prism, GraphPad, La Jolla, Calif.).

For antagonist mode experiments, a concentration curve (variableconcentration mode) or fixed amount (fixed concentration mode) ofantagonist was added to the cells, and allowed to equilibrate for 2minutes. Then, either a 5 nM fixed concentration (variable concentrationmode) or an 11 point concentration curve (fixed concentration mode) ofPACAP₁₋₂₇ was added to the cells, and the max-min response recorded asabove. For variable concentration mode experiments, the data wasnormalized to the stimulation caused by 5 nM PACAP₁₋₂₇ (100%) andvehicle (0%), and analyzed with a 3 variable non-linear curve fit, withthe IC₅₀ (nM) and I_(max) (%) calculated and reported (Prism). For thefixed concentration mode experiments, each curve was normalized to themaximum stimulation caused by PACAP₁₋₂₇ with no antagonist present(100%) and vehicle (0%). The resulting data was analyzed using aGaddum/Schild EC₅₀ shift model,(Schild, 1957, Gaddum, 1957) designed toanalyze competitive antagonism.(Lazareno and Birdsall, 1993) The dataoutput was the pA2 (nM) and the Schild Slope, a measure of how closelythe experimental data fits the operational model of competitiveantagonism (Prism). For all analyses, each independent experimentperformed in quadruplicate is considered to be a sample size of 1. Thepharmacology values are calculated separately from each experiment, thencombined and reported as the mean±SEM for the entire set of experiments.

The PC12 cells were cultured in RPMI containing 5% heat inactivatedfetal bovine serum and 10% horse serum in the presence of 100 units/mLpenicillin and 100 microgram/mL streptomycin. The cells were plated onpoly-D-Lysine coated 6-well tissue culture plates at a density of150,000 cells per well in 2 mL media. After 48 hours at 37° C. in 5% CO₂atmosphere, media exchange was performed and plates were dosed, usingthe peptide diluent (water) for the control samples. PACAP₁₋₂₇,PACAP_(1-27-S-G), and PACAP_(1-27-S-L) were used to screen for PAC₁receptor activation. Four groups of cells were used; one control group(diluent treated) and three treatment groups, each treatment group wasexposed to 100 nM concentrations of PACAP₁₋₂₇, PACAP_(1-27-S-G), orPACAP_(1-27-S-L). All groups were run in triplicate. Cell images of eachtreatment group were captured and compared to the control cells toscreen for differentiation and cell body volume increases. Cells havingneurite-like process outgrowth were noted and photographed. Theneurite-like outgrowth was deemed positive if its length was at leasttwo times the width of the cell body.

The foregoing discussion of the invention has been presented forpurposes of illustration and description. The foregoing is not intendedto limit the invention to the form or forms disclosed herein. Althoughthe description of the invention has included description of one or moreembodiments and certain variations and modifications, other variationsand modifications are within the scope of the invention, e.g., as may bewithin the skill and knowledge of those in the art, after understandingthe present disclosure. It is intended to obtain rights which includealternative embodiments to the extent permitted, including alternate,interchangeable and/or equivalent structures, functions, ranges or stepsto those claimed, whether or not such alternate, interchangeable and/orequivalent structures, functions, ranges or steps are disclosed herein,and without intending to publicly dedicate any patentable subjectmatter. All references cited herein are incorporated by reference intheir entirety.

1. A glycopeptide comprising a peptide that is covalently linked to asaccharide, wherein said peptide has from about 20 to about 40 aminoacid residues and at least 75% sequence identity to SEQ ID NO:1; SEQ IDNO:2 or SEQ ID NO:3, and wherein said saccharide comprises from 1 toabout 8 carbohydrates.
 2. The glycopeptide of claim 1, wherein saidpeptide is selected from the group consisting of PACAP₁₋₂₇, PACAP₁₋₃₈,VIP, [AC-His¹]PACAP-27, [Ala²]PACAP-27, [Gly²⁰]PACAP-27, andAc-[Phe(pI)⁶, Nle¹⁷]-PACAP₁₋₂₇.
 3. The glycopeptide of claim 2, whereinsaid [Ala²]PACAP-27 is [D-Ala²]PACAP-27.
 4. The glycopeptide of claim 1,wherein at least one of the amino acid residues of SEQ ID NOs:1, 2 or 3is substituted with a substitution amino acid residue having aside-chain functional group that is glycosylated.
 5. The glycopeptide ofclaim 4, wherein said substitution amino acid residue comprises serine,threonine, hydroxyproline or a similar ethanolamine linker.
 6. Theglycopeptide of claim 1, wherein the C-terminus end of said peptide isglycosylated.
 7. The glycopeptide of claim 1, wherein said glycopeptideis a pituitary adenylate cyclase-activating polypeptide type I receptor(PAC₁) agonist.
 8. The glycopeptide of claim 1, wherein saidglycopeptide is a VPAC₁ agonist.
 9. The glycopeptide of claim 1, whereinsaid glycopeptide is a selective PAC1 and VPAC1 agonist.
 10. Theglycopeptide of claim 1, wherein PAC1 binding affinity (Ki) of saidglycopeptide is less than about 10 nM.
 11. The glycopeptide of claim 1,wherein VPAC1 binding affinity (Ki) of said glycopeptide is less thanabout 10 nM.
 12. The glycopeptide of claim 1, wherein PAC1 agonistactivity (Ki) of said glycopeptide is less than about 10 nM.
 13. Theglycopeptide of claim 1, wherein VPAC1 agonist activity (κi) of saidglycopeptide is less than about 10 nM.
 14. The glycopeptide of claim 1,wherein said saccharide comprises from 1 to 3 carbohydrates.
 15. Theglycopeptide of claim 1, wherein said saccharide is a monosaccharide, adisaccharide.
 16. The glycopeptide of claim 1, wherein said peptidecomprises a plurality of glycosylated amino acid residues.
 17. Theglycopeptide of claim 1, wherein said saccharide is selected from thegroup consisting of glucose, maltose, lactose, melibiose, maltotriose,sucrose, trehalose, altose, saccharose, maltose, cellobiose, gentibiose,isomaltose, primeveose, galactose, xylose, mannose, manosaminic acid,fucose, GalNAc, GlcNAc, idose, iduronic acid, glucuronic acid, sialicacid, and polysaccharides related to the Thompsen-Friedrich antigens(Tn).
 18. The glycopeptide of claim 1, wherein said peptide comprises apeptide of SEQ ID NO:3.
 19. A method for treating a neurodegenerativedisease in a subject, said method comprising administering to thesubject in need of such a treatment a therapeutically effective amountof a glycopeptide of claim
 1. 20. The method of claim 19, wherein saidglycopeptide has a higher blood-brain barrier penetration compared tosaid peptide in the absence of said saccharide. 21-22. (canceled)